Isolation and molecular cloning of pneumolysin gene from group 19 pneumococci were studied to examine the relationship of pneumolysin to virulence. Group 19 chromosomal DNA samples were analyzed, using clonal ply genes from two different pneumococcal strains as probes. Genomic DNA that contained the ply gene from 19F, 19A, 19B and 19C strains were examined by polymerase chain reaction (PCR). The amplified gene contained a ClaI restriction enzyme site 5' and XbaI restriction enzyme site 3' encoded in the oligonucleotides utilized in the PCR mixture. The ClaI/XbaI ply gene was cloned into a vector, Bluescript pks(-). Recombinant clones 19A31 and 19F5 contained the ply gene as confirmed by restriction enzyme analysis and Southern blot. The nucleotide structure of 19A ply gene was sequenced and compared with type 2 ply gene. Ten differences in the two genes were observed in 1420 bp of sequence, whereas a change in only two amino acids (type 2 Arg226 -->19A Asp; type 2 Asp380 -->19A Asn) was present. Deduced amino acid sequence from 19A ply gene showed 24.6% homology with other cytolytic toxins, e.g. listeriolysin, alveolysin and perfringolysin.